Multi-Omics Atlas-Assisted Discovery of Transcription Factors for Selective T Cell State Programming

Abstract

CD8 ⁺ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection. To systematically define the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8 ⁺ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints of each state, uncovering new regulatory mechanisms that govern differentiation of selective cell states. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states critical in tumor and viral contexts: terminally exhausted T cells (TEX _term ), which are dysfunctional, and tissue-resident memory T cells (T _RM ), which are protective. Global TF community analysis revealed distinct state-specific biological pathways and TF-driven networks that govern protective versus dysfunctional T cell states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing ( in vivo Perturb-seq), we delineated TF-driven networks that govern protective versus dysfunctional T cell states. We identified HIC1 and GFI1 as shared regulators of TEX _term and T _RM differentiation and KLF6 as a unique regulator of T _RM . Importantly, we discovered novel TEX _term single-state TFs, including ZSCAN20 and JDP2 with no prior known function in T cells. Targeted deletion of these TFs enhanced tumor control and synergized with immune checkpoint blockade. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion-selective from protective T _RM programs, our platform enables more precise engineering of T cell states, advancing rational design of effective immunotherapies.